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PATHOGENETIC STUDIES IN JUVENILE
ESSENTIAL HYPERTENSION AND UREMIA
Role of oxidative stress and
gene polymorphisms of the renin-angiotensin system
Ph.D. thesis
Ferenc Papp M.D.
Consultants:
Sándor Túri M.D., D.Sc. and Csaba Bereczki M.D., Ph.D.
Department of Pediatrics,
Faculty of Medicine, University of Szeged
Szeged
2013
ii
List of publications Original papers directly related to the thesis I. F Papp, AL Friedman, Cs Bereczki, I Haszon, É Kiss, E Endreffy, S Túri. Renin-angiotensin gene polymorphism in children with uremia and essential hypertension. Pediatric Nephrology 2003; 18:150–154. [IF: 1.219] II. S Túri, A Friedman, Cs Bereczki, F Papp, J Kovács, E Karg, I Németh. Oxidative stress in juvenile essential hypertension. Journal of Hypertension 2003; 21:145–152. [IF: 3.572] Other papers related to the topic of the thesis Cs Bereczki, S Túri, I Németh, É Sallai, Cs Torday, E Nagy, I Haszon, F Papp. The roles of platelet function, thromboxane, blood lipids and nitric oxide in hypertension of children and adolescents. Prostaglandins, Leukotrienes and Essential Fatty Acids 2000; 62:293–297. [IF: 1.226] I Haszon, AL Friedman, F Papp, Cs Bereczki, S Baji, T Bodrogi, É Károly, E Endreffy, S Túri. ACE gene polymorphism and renal scarring in primary vesicoureteric reflux. Pediatric Nephrology 2002; 17:1027–1031. [IF: 1.420] I Haszon, F Papp, M Bors, Cs Bereczki, S Túri. Platelet aggregation, blood viscosity and serum lipids in hypertensive and obese children. European Journal of Pediatrics 2003; 162:385–390. [IF: 1.157] Túri S, Haszon I, Papp F, Bereczki Cs, Endreffy E, Németh I. A juvenilis esszenciális hypertonia patomechanizmusa. Hypertonia és Nephrologia 2001; 5:131–136. Papp F, Haszon I, Kovács J, Bors M, Németh I, Bereczki Cs, Túri S. Thrombocyta aggregáció, vér viszkozitás és szérum lipidek alakulása hipertóniás és obes gyermekekben. Transzfúzió 2004; 37:3–12.
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Table of contents
List of publications ii
Abbreviations v
1. INTRODUCTION 1
1.1. Epidemiology of essential hypertension and obesity in children 1
1.2.
Pathogenetic factors in essential hypertension and obesity-induced hypertension
2
1.2.1. Oxidative stress 4
1.2.2. Oxidative stress in hypertension and obesity 5
1.2.3. Endothelial dysfunction 7
1.2.4.
Genetic aspects of essential hypertension, gene polymorphisms of the renin-angiotensin system 11
1.2.5.
Possible age-related differences in the pathogenesis of essential hypertension 13
1.3. Renal fibrosis, the role of angiotensin II 13
2. AIMS AND QUESTIONS OF THE STUDIES 16
3. PATIENTS 18
3.1. Clinical characteristics of the oxidative stress study population 18
3.2.
Clinical characteristics of subjects in the genetic study of renin-angiotensin system
20
4. METHODS 22
4.1. Biochemical methods 22
4.2. Determination of the renin-angiotensin system gene polymorphisms 24
4.3. Statistical analysis 26
iv
5. RESULTS 27
6. DISCUSSION 35
6.1.
Oxidative stress and endothelial dysfunction in juvenile essential hypertension and obesity 35
6.2.
Gene polymorphisms of the renin-angiotensin system in juvenile essential hypertension and uremia 37
7. SUMMARY OF OUR FINDINGS AND CONCLUSIONS 42
8. ACKNOWLEDGEMENTS 44
9. REFERENCES 45
Appendix 53
v
Abbreviations
Ang II angiotensin II
ACE angiotensin converting enzyme
ADPKD autosomal dominant polycystic kidney disease
APH acetylphenylhydrazine
AT1R angiotensin II type 1 receptor
AGT angiotensinogen gene
AGTR1 angiotensin II type 1 receptor gene
BH4 tetrahydrobiopterin
bp base pair
BP blood pressure
BMI body mass index
CAT catalase
CRF chronic renal failure
CV coefficient of variation
DBP diastolic blood pressure
DNA deoxyribonucleic acid
ECM extracellular matrix
EH essential hypertension
eNOS endothelial nitric oxide synthase
ESRD end stage renal disease
ET-1 endothelin-1
FSGS focal segmental glomerulosclerosis
GPx glutathione peroxidase
GSH reduced glutathione
GSSG oxidized glutathione
H2O2 hydrogene peroxide
vi
Hb hemoglobin
LPO lipid peroxidation
LVH left ventricular hypertrophy
MDA malondialdehyde
NADPH nicotinamide adenine dinucleotide phosphate
NO nitric oxide
NOS nitric oxide synthase
NOx sum of nitrite and nitrate
O2 ̄ superoxide
OH obesity-induced hypertension
OR odds ratio
PCR polymerase chain reaction
RAS renin-angiotensin system
RBC red blood cells
ROS reactive oxygen species
SBP systolic blood pressure
SOD superoxide dismutase
SNS sympathetic nervous system
TGF- transforming growth factor-
TxA2 thromboxane A2
VSMC vascular smooth muscle cells
XO xanthine oxidase
1
1. INTRODUCTION
1.1. Epidemiology of essential hypertension and obesity in children
Essential hypertension (EH) is one of the most common disorders in adulthood and is the
leading cause of premature death among adults [1]. Previously it was believed that secondary
hypertension is the major form of childhood hypertension, but the clinical importance of
primary or EH in children, and especially in adolescents, has significantly increased in the last
decade which can be observed in the epidemiologic data. The increasing prevalence of
childhood high blood pressure (BP) has been reported in several studies and national surveys.
In the past, the estimated prevalence of childhood hypertension was 1–2%. Recent reports
indicate that hypertension now affects 3–5% of the whole pediatric population, and at least the
same percentage of children is in pre-hypertensive stage [2–6]. In a Hungarian population of
adolescents the prevalence of hypertension was 2.53% [7]. The global public health
significance of high BP in childhood as well as adolescence is based on observations that
confirm a strong tracking of BP levels from childhood to adulthood. Children with high
normal BP during adolescence have a greater tendency to develop hypertension during
adulthood [8]. Over the last decade many studies have provided supporting evidence that
hypertension associated vascular damage also develops in childhood, and many of the
hypertensive children do in fact present with hypertensive end organ damage such as left
ventricular hypertrophy (LVH) [9].
The increasing prevalence of high BP in children and adolescents is largely due to the
increase in prevalence of obesity all over the world. In the United States childhood obesity
has more than doubled in children and tripled in adolescents in the past 30 years, and now
affects nearly 20% of the whole pediatric population. In 2010, more than one third of the
children and adolescents were overweight or obese [10]. According to the WHO European
Childhood Obesity Surveillance Initiative 2008 report the same trend can be seen in Europe.
Data of 6–9-year-old children from 12 European countries were collected. The prevalence of
overweight (including obesity) ranged from 18.4% to 49%, and 4.6 to 26.6% of children were
actually obese [11].
2
In a large cross sectional survey performed in the period 2005–2006 the overall prevalence of
overweight and obesity among the Hungarian schoolchildren aged 11–16 years was found to
be 23.4% and 6.6% of the subjects were obese. Between the years 2001 and 2006, the
prevalence of overweight and obesity nearly doubled from 13% to 23.4% in this age group of
Hungarian adolescents [12].
Obesity is the primary risk for EH in children. The relationship between obesity and
hypertension in children has been reported in numerous studies targeting a variety of ethnic
and racial groups. All of these studies found higher BP and/or higher prevalence of
hypertension in obese compared with lean controls [13]. Sorof et al. reported that more than
one third of obese school children have abnormally elevated BP [5]. Apart from hypertension
obesity is associated with other cardiovascular risk factors, such as dyslipidemia, insulin
resistance, glucose intolerance, type 2 diabetes mellitus, LVH, and pulmonary hypertension.
Many of these outcomes of obesity may begin in childhood and adolescence [13].
1.2. Pathogenetic factors in essential hypertension and obesity-induced hypertension
Human EH is a multifactorial disorder, and many pathophysiological factors have been
implicated in its genesis (Table 1) [8, 14].
Obesity-induced hypertension or obesity hypertension (OH) is often considered a
special form of hypertension, but considerable evidence indicates that obesity is the most
common cause of EH. Hypertension and other obesity-related cardiovascular complications
are associated with visceral/abdominal adiposity. OH is a complex and multifactorial
condition. The pathophysiologic mechanisms whereby obesity causes hypertension have not
been fully elucidated. Abnormal kidney function is an important cause as well as consequence
of OH. Increased renal tubular sodium reabsorption and impaired pressure natriuresis play key
roles in the initiation of hypertension. Several mechanisms contribute to altered kidney
function and hypertension in obesity, but three of these mechanisms are especially important:
1) increased activity of the SNS, 2) the activation of the RAS, and 3) physical compression of
the kidneys by fat accumulation within and around the kidneys and by increased abdominal
pressure due to visceral fat. The activation of SNS appears to be mediated partly by the
increased levels of the adipocyte-derived hormone leptin, and the activation of the
3
hypothalamic leptin – pro-opiomelanocortin – melanocortin 4 receptor pathway.
Inflammatory cytokines and free fatty acids released from adipocytes, angiotensin II (Ang II),
and other factors have also been suggested as mediators of SNS activation in obesity. In
addition, hyperinsulinemia and/or insulin resistance and dyslipidemia, impaired endothelial
function, genetic and lifestyle factors may also be of significance in the pathophysiology of
OH [15–18].
Table 1. Pathogenetic factors of human essential hypertension
increased sympathetic nervous system activity
overproduction of sodium-retaining hormones and vasoconstrictors
long-term high sodium intake
increased or inappropriate renin secretion with resultant increased production of angiotensin II and aldosterone
deficiencies of vasodilators, such as nitric oxide, prostacyclin, and the natriuretic peptides and/or overproduction of vasoconstrictor endothelin-1
alterations in expression of the kallikrein-kinin system
abnormalities of resistance vessels
increased activity of vascular growth factors
alterations in adrenergic receptors
altered cellular ion transport
structural and functional abnormalities in the vasculature, including endothelial dysfunction, and vascular remodeling
oxidative stress
hyperuricemia
insulin resistance and diabetes mellitus
obesity
genetic factors
fetal programming
Hereinafter, the pathogenetic factors of EH and OH related to our investigations will be
discussed, namely oxidative stress, endothelial dysfunction and genetic predisposing factors.
4
1.2.1. Oxidative stress
In the course of normal aerobe cellular metabolism several highly reactive molecules are
generated. These reactive species are generally reactive oxygen species (ROS), such as
superoxide (O2̄ ), hydrogen peroxide (H2O2) and hydroxyl radical, as well as reactive nitrogen
species, e.g. peroxynitrite, which result from the cellular redox process and many of them are
free radicals. The primary sources of ROS include mitochondrial electron transport system
and various oxidase enzymes in the cell, and uncoupled nitric oxide synthase (NOS) among
others. Moreover, free radicals can be derived from exogenous sources such as environmental
toxins and cigarette smoke. It is important to note that while the excessive production of ROS
causes injury and dysfunction, the normal rate of ROS production is essential for various
physiological processes, such as gene expression, signal transduction, regulation of cell
growth and apoptosis, fetal development, and innate immunity [19–21]. Under normal
conditions, ROS and the byproducts of their reactions with various biomolecules are
converted to harmless molecules by the natural antioxidant system [20]. The antioxidant
defense system is a highly complex biochemical organization which consists of numerous
enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase
(GPx), and a large number of scavenger molecules, e.g. ascorbic acid (Vitamin C), alpha-
tocopherol (Vitamin E), glutathione, flavonoids, urate, and the like [21].
Oxidative stress is characterized by the imbalance between ROS production and
antioxidant defense capacity of the body. This can be the result of either increased ROS
generation, impaired antioxidant system, or the combination of both [20]. ROS are capable of
damaging all types of biomolecules, including nucleic acids, proteins, and lipids. DNA
modification by ROS may be the initial step in mutagenesis, carcinogenesis and aging.
Proteins may also be damaged by ROS thus leading to structural changes and loss of enzyme
activity. Oxidative degradation of lipids, called lipid peroxidation (LPO) affects many cellular
components, but the primary action-sites involve membrane associated polyunsaturated fatty
acids. The peroxidation of membrane associated fatty acids and cholesterol can alter cell
membrane fluidity and permeability, and may eventually induce membrane damage. LPO also
changes the low density lipoprotein to proatherogenic and proinflammatory forms [19, 22,
23].
5
Further decomposition of peroxidized lipids results in a wide variety of harmful end-products,
such as malondialdehyde (MDA) and F2-isoprostanes. These products of LPO have
commonly been used to assess oxidative stress in vivo [23, 24].
Oxidative stress and ROS attack modify and denature functional and structural
molecules thus leading to tissue injury and dysfunction. Oxidative stress plays a role in
inflammation, accelerates aging and contributes to a variety of chronic and degenerative
conditions, such as cancer, diabetes, autoimmune disorders, inflammatory diseases,
rheumatoid arthritis, respiratory and kidney diseases, atherosclerosis, cardiovascular and
neurodegenerative diseases [21, 22].
1.2.2. Oxidative stress in hypertension and obesity
Oxidative stress in hypertension
The association between oxidative stress and hypertension has been extensively studied. Most
of the reports in animals as well as in humans demonstrate an increased oxidative stress in
hypertension. Vascular oxidative stress has been observed in spontaneously hypertensive rats
and various experimental animal models of hypertension. Increased ROS release from
isolated vessels of hypertensive animals has also been detected [25–27]. There is extensive
evidence concerning the increase of oxidative stress in human EH. Most of the studies using
non-specific markers of oxidative damage demonstrate an obvious connection between
oxidative stress and hypertension. Higher O2 ̄ and H2O2 production have been observed in
hypertensive subjects, which returned to levels observed in control subjects after BP
reduction. Increased LPO, and an imbalance in antioxidant status was reported in
hypertensive patients, suggesting that oxidative stress is important in the pathogenesis of EH.
Moreover, decrease in SOD and GPx activities have been demonstrated in newly diagnosed
and untreated hypertensive subjects, and SOD activity was inversely correlated with BP
within the hypertensive group [26]. In a study by Rodrigo et al., daytime systolic BP (SBP)
and diastolic BP (DBP) of hypertensive patients negatively correlated with plasma antioxidant
capacity, plasma vitamin C levels, erythrocyte activity of antioxidant enzymes (SOD, CAT,
GPx), and erythrocyte reduced/oxidized glutathione ratio, showing a higher level of oxidative
stress in hypertensive subjects. They also reported that BP positively correlated with both
6
plasma and urine 8-isoprostane (8-iso-prostaglandin F2α) [28]. F2-isoprostanes are considered
the best available biomarkers of oxidative stress status and LPO in vivo [24]. In studies using
these highly specific markers the relationship between oxidative stress and hypertension is not
convincing. The urinary concentrations of F2-isoprostane were found to be the same in
subjects with mild to moderate untreated hypertension as well as in the normotensive control
group. In another study no difference has been observed in plasma and 24-h urinary F2-
isoprostanes in treated or untreated hypertensive subjects compared with normotensive
controls [26].
The casual role of oxidative stress in hypertension is supported by several observations.
Some studies in animal models of hypertension showed that the administration of
pharmacological doses of antioxidants may reduce BP [20]. However, the data from clinical
trials using antioxidants are less convincing, and most of them failed to show that the
supplementation of antioxidants reduces BP [26, 27]. Induction of oxidative stress has been
shown to cause hypertension in normal animals. Binding of Ang II to its type 1 receptor
(AT1R), results in ROS production via activation of NADPH oxidase in the kidney and
vasculature. The ROS production and hypertensive response to Ang II infusion is attenuated
by pharmacological inhibition of NADPH oxidase. These observations demonstrate the role
of ROS as a major mediator of the pressor action of Ang II [20]. Oxidative stress may
contribute to the generation and/or maintenance of hypertension by several mechanisms,
including damage to endothelial cells and endothelial dysfunction, damage to vascular smooth
muscle cells (VSMC), generation of vasoconstrictor LPO products, stimulation of
inflammation and growth signaling events [26].
Oxidative stress may not only be the cause, but also the consequence of hypertension.
Animal experiments clearly illustrate the role of high BP and shear stress in the generation of
vascular oxidative stress [20].
Although excessive production of ROS is the most common cause of oxidative stress in
hypertension, it is occasionally caused by the primary impairment of the antioxidant system as
it is seen in SOD deficient, therefore hypertensive mice. Moreover, the consumption of
antioxidant molecules and the inactivation of the antioxidant enzymes caused by permanent
oxidative stress can impair the antioxidant defense system and can enhance oxidative stress
itself [20].
7
Oxidative stress in obesity
Several studies provide evidence that obesity per se is associated with enhanced oxidative
stress. Keaney et al. reported that a relation between increasing BMI and increasing systemic
oxidant stress may be observed. Using the quantification of urinary F2-isoprostanes, it is
shown in nearly 3000 patients involved in the Framingham Heart Study that enhanced
isoprostane formation is strongly associated with increasing body mass index (BMI) [29]. In a
study by Furukawa et al., fat accumulation closely correlated with the plasma and urinary
LPO end-products. In addition, it was demonstrated that plasma adiponectin levels correlated
inversely with oxidative stress markers. These results suggest that fat accumulation itself
could increase systemic oxidative stress, and that increased oxidative stress in obesity might
relate to the dysregulated production of adipocytokines [30].
Multiple mechanisms may contribute to increased oxidative stress in obesity. A number
of ROS producing pathways are known to be perturbed in obesity. For example, RAS is
activated, and Ang II induces ROS production via the activation of NADPH oxidase, and it
also enhances lipoprotein oxidation. Moreover, obesity is also associated with reduced
antioxidant defense mechanisms, elevated systemic inflammation and activation of
coagulation cascades [31, 32]. The chronic over-nutrition in obesity itself can induce
oxidative stress. High caloric intake composed of glucose, lipid or protein causes an increase
in the generation of ROS by leukocytes. Lipid intake causes a prolonged increase in LPO
[26]. On the other hand, caloric restriction in the obese and fasting in normal subjects leads to
a marked reduction in ROS generation by leukocytes and other indexes of oxidative stress
[33].
1.2.3. Endothelial dysfunction
The endothelium plays a fundamental role in the regulation of vascular function as it produces
a large number of biologically active agents that participate in the regulation of vascular tone,
cell growth, inflammation, and thrombosis/hemostasis [34]. Vasoactive substances derived
from endothelium are vasodilators, among others nitric oxide (NO), and prostacyclin, or
vasoconstrictors, such as endothelin-1 (ET-1), Ang II, thromboxane A2 (TxA2), and ROS.
8
Other inflammatory modulators, adhesion molecules, chemokines, and factors with
hemostatic properties are also released by endothelium [35].
Primarily, endothelial dysfunction is characterized by impaired vasodilation to specific
stimuli or an imbalance between endothelium derived vasodilation and vasoconstriction,
resulting in reduced vasodilation [35–37]. In addition, it is also associated with a
proinflammatory and prothrombic state. The dysfunction of the vascular endothelium has
been implicated in the pathophysiology of various cardiovascular diseases, including
hypertension, coronary artery disease, chronic heart failure, peripheral artery disease,
diabetes, and chronic renal failure [35].
Multiple pathways are involved in the development of endothelial dysfunction; the main
are: reduced NO, oxidative stress, vasoactive peptides, and inflammation. NO is the key
endothelium-derived vasodilator that plays a pivotal role in the maintenance of vascular tone
and reactivity, and also opposes the actions of endothelium-derived vasoconstrictors.
Furthermore, it inhibits growth, inflammation, and aggregation of platelets [35, 36]. Not
surprisingly, that reduced NO in the vessel wall has a central role in the complex mechanism
of endothelial dysfunction. The reduction in NO may result from reduced endothelial NOS
activity (eNOS; the enzyme, which synthesizes NO from L-arginine, with the cofactor
tetrahydrobiopterin [BH4] in the endothelium) and/or decreased NO bioavailability [35].
Oxidative excess may cause NO reduction by different mechanisms. Probably the most
important is the inactivation of NO by ROS, which diminishes the amount of available NO,
while a cytotoxic oxidant, peroxynitrite is formed. Furthermore, in oxidative states, the
reduction in BH4 caused by ROS and peroxynitrite results in uncoupling of eNOS, which
leads to further ROS formation [35, 37]. The main sources of ROS implicated in the genesis
of endothelial dysfunction are NADPH oxidase, uncoupled eNOS, and xanthine oxidase (XO)
[38]. Moreover, oxidative stress induces proinflammatory and prothrombic state, and it is also
involved in the detachment and apoptosis of endothelial cells [35]. A relation between
oxidative stress and endothelial dysfunction, characterized by decreased NO has been
demonstrated in animal models as well as humans with hypertension and chronic renal failure
[39]. The oxidized end-products of NO are nitrite and nitrate, and the sum of their plasma
levels (NOx) are frequently used markers of NO bioavailability and endothelial function in
vivo [40, 41].
9
Vasoconstrictors, such as Ang II and ET-1 also have been implicated in the development of
hypertension and endothelial dysfunction. Pathogenic effects of these substances are partially
mediated by ROS production due to the activation of NADPH oxidase [34, 42]. Our previous
studies allude to the importance of vasoactive components in childhood EH. Increased in vitro
platelet aggregation and thromboxane B2 (stable degradation product of TxA2) levels were
observed in hypertensive children [43]. In a second study, the increased platelet aggregability
was also confirmed in non-obese and obese hypertensive children, and simultaneously,
decreased NO and depletion of plasma free thiols, – as the sign of oxidant injury – were
demonstrated in both groups of hypertensives [44]. Increase in platelet aggregation releases
TxA2, serotonin, and other mediators causing local vasoconstriction and further aggregation.
The major pathways of endothelial dysfunction focusing on the role of oxidative stress
and NO are summarized in Figure 1. The investigated markers of oxidative stress and
endothelial dysfunction in our study are also illustrated. O2 ̄ is generated by different enzymes
in the endothelial cells; the main are the NADPH oxidase, uncoupled eNOS and XO. NO is
synthetized from L-arginine by eNOS. The action of different vasoconstrictive factors, such as
Ang II, ET-1, cytokines, mechanical stretch is mainly mediated by the activation of NADPH
oxidase leading to O2 ̄ production, which can causes the constriction of VSMC, leads to further
vasoconstrictive peptide production (Ang II, ET-1) and inactivates NO resulting in
peroxynitrite formation. Peroxynitrite and oxidative excess lead to the uncoupling of eNOS.
The uncoupled eNOS has reductase function and produces ROS instead of NO, and
aggravates the oxidative damage. Reduction in NO caused by decreased eNOS activity (due
to ET-1 among others) and inactivation of NO by ROS results in impaired vasodilation,
increased platelet aggregation, and amplifies the inflammatory process. From aggregated
platelets TxA2, and other mediators are released causing vasoconstriction and further
aggregation. Oxidative stress induces proinflammatory and prothrombic state by the
upregulation of different endothelium-derived cytokines, chemokines and hemostatic factors,
leads to LPO and MDA production, and also enhances platelet aggregation. The imbalance
between vasoconstrictors and vasodilators, mainly between ROS as vasoconstrictors and NO
as vasodilator is a fundamental feature of endothelial dysfunction. Antioxidant mechanisms
are activated in the presence of oxidative stress; oxidized glutathione (GSSG) is formed from
reduced glutathione (GSH), O2 ̄ is transformed to H2O2 by SOD, which is neutralized by CAT
10
or GPx, and reduced plasma thiols (-SH) will be oxidized (-S-S-). Red blood cells (RBC)
circulate in the direct vicinity of the endothelial surface. Their membranes are permeable to
oxidants, therefore, antioxidants of the RBC are also activated. Decrease in end-products of
NO metabolism (NOx) is correlated to NO reduction in endothelial dysfunction.
Figure 1. Schematic figure of the major pathways of endothelial dysfunction. See the text for the
details. NO, nitric oxide; O2̄, superoxide; eNOS, endothelial nitric oxide synthase; XO, xanthine
oxidase; Ang II, angiotensin II; ET-1, endothelin-1; VSMC, vascular smooth muscle cells; ROS,
reactive oxygen species; TxA2, thromboxane A2; LPO, lipid peroxidation; MDA, malondialdehyde;
GSSG, oxidized glutathione; GSH, reduced glutathione; H2O2, hydrogen-peroxide; SOD, superoxide
dismutase; CAT, catalase; GPx, glutathione peroxidase; -SH, reduced plasma thiols; -S-S-, oxidized
plasma thiols; RBC, red blood cells; NOx, NO end-products.
11
1.2.4. Genetic aspects of essential hypertension, gene polymorphisms of the renin-
angiotensin system
BP level is a complex trait resulting from both genetic and environmental factors. It is widely
accepted, that approximately 30–60% of the phenotypic variation in BP is determined by
genetic factors [45]. The common form of EH is a polygenic disorder. It means that the
development of hypertensive phenotype results from the effects of multiple genes and is
modulated by multiple environmental factors. There are two approaches for the genetic
dissections of complex and quantitative traits, such as EH: genome-wide scanning and
candidate gene approach, and both have specific advantages and disadvantages [46]. Most
published data on human EH arise from candidate gene studies. Many candidate genes have
been tested for association with BP and hypertension in case-control and sib pair linkage
studies with conflicting results.
Gene polymorphisms of the renin-angiotensin system
Polymorphisms in genes of the RAS have been the most commonly studied as potential
genetic risk factors for hypertension and other cardiovascular diseases. The RAS is a
multienzyme, multilocale hormone system, which regulates blood pressure, as well as the
fluid and electrolyte balance, and also plays essential roles in several physiological and
pathological processes. Two forms of the RAS exist, the systemic/endocrine and local/tissue
RAS. The cascade of the RAS consists of two enzymatic steps: angiotensin I is cleaved from
angiotensinogen by the action of renin, which undergoes a second cleavage, mainly by tissue
angiotensin converting enzyme (ACE) to generate Ang II, the main effector hormone of the
system, which acts primarily on AT1R resulting in aldosterone secretion from the adrenal
cortex and many other systemic and/or locally effects. Because the RAS is intimately
involved in the regulation of BP, a genetic variability in the degree of expression of its
proteins may account for variability in the BP or may play a role in mediating hypertension
[47].
The angiotensinogen gene (AGT) is located on 1q42, and consists of 5 exons. Molecular
variant of AGT gene encoding threonine (T) instead of methionine (M) at position 235
(M235T) in exon 2 has an influence on the angiotensinogen level. The T235 allele is
12
associated with higher angiotensinogen levels, which could theoretically translate into higher
Ang II levels and may lead to hypertension and other cardiovascular complications [48, 49].
The ACE gene is located on 17q23, and comprises 26 exons. The ACE gene
insertion/deletion (I/D) polymorphism is characterized by the presence (insertion) or absence
(deletion) of a 287-bp AluI-repeat sequence inside intron 16. I/D polymorphism is strongly
associated with variations in circulating and tissue ACE levels [47, 50–52]. The DD genotype
with higher tissue ACE levels would correspond to higher Ang II generation in the vascular
tissue leading to hypertension, and pathological cardiovascular states.
Most of the known actions of Ang II are mediated by the AT1R. The AT1R gene
(AGTR1) maps to 3q24, containing five exons. Several polymorphisms of the AT1R gene
have been identified. Polymorphism substituting cytosine (C) for adenine (A) at position
+1166 in the 3’-untranslated region (A1166C) was the only, which had significantly increased
frequency in hypertensive individuals. The physiological significance of this polymorphism is
uncertain [53].
The possible pathogenetic effects of gene polymorphisms of the RAS in hypertension
are presented and discussed in the Discussion chapter.
Other candidate genes for association with essential hypertension
Aldosterone, a major effector hormone of the systemic RAS, regulates the sodium and
potassium balance in the kidney, thus plays a fundamental role in the regulation of
extracellular volume. Gene encoding aldosterone synthase (CYP11B2) has also emerged as a
candidate gene for hypertension [54]. Variations and mutations in a huge number of other
candidate genes, such as α-adducin, eNOS, ET-1, atrial natriuretic factor, β2-adrenergic
receptor, insulin receptor, and many others, have also been reported to be associated with EH
[8, 54]. For example, in a study published by our group, polymorphism of the ET-1 gene
(G5665T) was significantly associated with EH and particularly OH. The same association
was not detected in case of eNOS polymorphisms (T-786C promoter polymorphism and 4th
intron 27-bp repeat polymorphism) [55].
13
1.2.5. Possible age-related differences in the pathogenesis of essential hypertension
The pathogenesis of EH in children and adolescents bears a close resemblance to that of the
adults. The minor variations observed could mostly be due to the evolving nature of this
condition [8]. On the other hand, most of the pathogenetic studies, including genetic
association studies, have been performed in different hypertensive groups of adults.
Therefore, studies in the pediatric and adolescent age groups may help to point out the
possible age-related differences in the pathogenesis of essential hypertension. According to
our supposition the effects of certain pathogenetic factors of EH compared to adults may be
stronger or weaker in children. Children are relatively free from the common environmental
and other risk factors (alcohol consumption, smoking, diabetes mellitus, dyslipidemia)
contributing to hypertension. Therefore, we presume that the role of oxidative stress,
endothelial dysfunction, and genetic predisposing factors may be more important in the
occurrence of hypertension in children than in adults. In addition, lack of environmental and
cardiovascular risk factors which are mainly associated with hypertension in adulthood allows
a more precise investigation of oxidative stress and endothelial dysfunction in the case of
hypertension of children and the alterations reflect hypertension only.
1.3. Renal fibrosis, the role of angiotensin II
Renal fibrosis or scarring, characterized by glomerulosclerosis and tubulointerstitial fibrosis,
is the final common manifestation of a wide variety of chronic kidney diseases, irrespectively
of the initial causes. The pathogenesis of renal fibrosis is a progressive process that clinically
leads to chronic renal failure (CRF), and ultimately to end stage renal disease (ESRD), a
devastating condition that requires dialysis or kidney transplantation [56].
Renal fibrosis represents a failed wound-healing response of the kidney tissue after
chronic, sustained injury, which leads to the excessive deposition of the extracellular matrix
(ECM) components. Briefly, activated kidney resident cells produce proinflammatory
cytokines, and for this signal inflammatory monocytes/macrophages and T cells infiltrate the
injured sites. Glomerular or interstitial infiltrated inflammatory cells become activated and
produce injurious molecules, such as ROS, as well as fibrogenic and inflammatory cytokines.
14
Then, fibrogenic or fibrosis-promoting factors activate the matrix producing effector cells
(glomerular mesangial cells, interstitial fibroblasts, and tubular epithelial cells, depending on
the nature and sites of injury) to produce interstitial matrix components. Several different
fibrogenic factors have been documented, but primary roles suggested transforming growth
factor-β (TGF-β), connective tissue growth factor, Ang II and ET-1. Among them TGF-β has
the highest significance, its induction appears to be a convergent pathway, that integrates
directly or indirectly the effects of many other fibrogenic factors. The activated cells produce
a large amount of ECM components; they are deposited in the extracellular compartment, and
are often crosslinked and become resistant to degradation. Defective matrix degradation
processes also contribute to ECM accumulation. Continuous deposition of ECM proteins
begins to have destructive effects on the kidney structure, fibrous scars are generated, leading
to the collapse of renal parenchyma and the loss of kidney function [56–58].
Angiotensin II in renal fibrosis
The role of Ang II in the pathogenesis of progressive kidney diseases is well established. In
experimental models of kidney damage renal RAS activation, cell proliferation, growth factor
upregulation and matrix production have been observed. Treatment with blockers of Ang II
actions prevents proteinuria, inflammatory cell infiltration and fibrosis, and retards disease
progression. Ang II could be involved in the fibrotic process in different ways. Ang II is not
only a vasoactive agent, it is also a renal growth factor that activates ECM producing cells,
increasing the expression and synthesis of ECM proteins, leading to glomerulosclerosis and
tubulointerstitial fibrosis. These effects seem to be mediated mainly by TGF-β. Ang II has
been shown to stimulate the TGF-β production of various cells, including renal tubular cells
and fibroblasts. The use of ACE inhibitors or AT1R antagonists in experimental models of
renal diseases reduces TGF-β production and attenuates renal fibrosis. In addition, Ang II
activates mononuclear cells and increases the production of proinflammatory mediators
(cytokines, chemokines, adhesion molecules), decreases matrix degradation, and may have
direct effect on collagen gene expression. Ang II is also capable of affecting renal
hemodynamics, increasing glomerular pressure and proteinuria, and enhancing
tubulointerstitial ischemia, which may contribute to fibrotic process [59, 60].
15
Gene polymorphisms of the RAS affect the tissue Ang II level in the kidney, and therefore
may have an influence on renal fibrogenesis [61]. This presumption has been studied in
different types of chronic kidney diseases, mainly in adults, but also in children. Some of
them showed a significant relation between genetic variants of the RAS and progressive loss
of renal function. Genetic association studies of the RAS gene polymorphisms in chronic
renal diseases will be reviewed in the Discussion chapter.
16
2. AIMS AND QUESTIONS OF THE STUDIES
The aims of our investigations were 1) to recognize the presence of oxidative stress and the
related endothelial dysfunction in EH of adolescents (juvenile EH), BMI being taken into
consideration as a confounding factor, and 2) to determine the polymorphisms of the RAS
genes in adolescents with EH and patients with uremia. Thus, assessments were made of the
relationship between NO production via its end-products in the plasma (nitrite + nitrate, NOx)
and the usual indicators of oxidative stress: the levels of plasma peroxidation end-products
and free thiols. As a new parameter, the redox status of glutathione, and the glutathione
recycling capacity of the RBC were also measured in patients with juvenile EH compared
with normotensive controls with a similar BMI. In the genetic association study adolescents
with EH, and also pediatric and adult patients with ESRD were genotyped for the M235T
polymorphism of AGT gene, the I/D polymorphism of ACE gene and the A1166C
polymorphism of AT1R gene.
The main questions of the studies were the following:
Question 1.
Is there any relationship between blood pressure and body mass index?
Question 2.
Does nitric oxide production, which is characterized by plasma concentration of its end-
products (nitrite + nitrate), decrease in juvenile essential hypertension? Does it depend on
body mass index?
Question 3.
Can we observe an increased oxidative stress state in juvenile essential hypertension? Is there
any correlation between oxidative stress and blood pressure? Does the oxidative stress
correlate with body mass index in normotensive and hypertensive adolescents?
17
Question 4.
May the polymorphisms of the renin-angiotensin system genes have any influence on the
development of juvenile essential hypertension?
Question 5.
Is there any role of the renin-angiotensin system gene polymorphisms in the development of
end stage renal disease in children and adults?
18
3. PATIENTS
3.1. Clinical characteristics of the oxidative stress study population
Fifty-two hypertensive patients (mean age 14.4 3.1 years, male/female 37/15) and
simultaneously, 48 age-matched control subjects (mean age 14.3 4.3 years, male/female
20/28) with normal BP were studied for the biochemical analysis of oxidative stress.
Hypertension was defined as a BP greater than the 95th percentile for age, gender and
height on three separate occasions at 5-min intervals at the time of enrolment [62]. Thereafter,
the diagnosis of hypertension was confirmed with ambulatory BP monitoring as 24-h SBP
and/or DBP mean values are equal to or greater than the 95th percentile of the age, height, and
sex-matched normal values [63]. An oscillometric ambulatory BP monitor (ABPM-04;
Meditech Kft., Budapest, Hungary) was used to record 24-h BP in hypertensive patients. BP
was registered automatically at 15-min intervals during the day and 30-min intervals during
the night. The measured values were analyzed by a computer (Medibase software) and shown
as 24-h systolic and diastolic means. Cases of endocrine, cardiological, neurological, renal,
and renovascular origin were excluded. Neither proteinuria (defined as > 300 mg/24-h urine)
nor any impairment in renal function (creatinine clearance < 80 ml/min per 1.73 m2) was
observed. The patients had not yet received any treatment. They kept a regular diet, with a
similar caloric intake and physical activity as the controls. None of them were smokers, as
controlled via the carbon monoxide hemoglobin concentrations. Blood samples were always
collected at the same time of the day (at 9:00 h). Twenty children were the offspring of treated
hypertensive parents.
The BMI (weight in kilograms divided by the square of the height in meters) was used
as a measure of ponderosity. According to their BMI both the hypertensive and the control
groups were divided into normal (< 25 kg/m2) and overweight (> 25 kg/m2) subgroups.
However, in order to acquire more information about the potential relation between the
weight status and the oxidative stress parameters, the results of the biochemical examinations
were analyzed and are also listed in the figures, as lean (BMI < 20 kg/m2), normal
(BMI = 20–25 kg/m2), overweight (BMI = 25–30 kg/m2) and obese (BMI > 30 kg/m2) groups.
19
Demographic data, physical characteristics and some metabolic parameters of the biochemical
study population are presented in Table 2. The concentrations of metabolic parameters
(plasma glucose, cholesterol and triglyceride) revealed similar increase in overweight/obese
subjects, both in the controls and in the patients with hypertension. Naturally, the BP values
were significantly higher in hypertensive patients as compared with their appropriate BMI
control groups (Table 2).
Table 2. Demographic data, physical characteristics and some metabolic parameters of
the biochemical study population, related to their body mass index (BMI, kg/m2)
Controls (n = 48)
Hypertensive patients (n = 52)
BMI < 25 (n = 26)
BMI > 25 (n = 22)
BMI < 25 (n = 28)
BMI > 25 (n = 24)
Age (years) 14.6 4.6 14.4 5.1 14.4 3.1 14.4 2.5
Sex (male/female) 13/13 14/8 18/10 19/5
Body weight (kg) 51 9 81 12 54 13 86 11
Height (m) 1.60 0.18 1.62 0.12 1.65 0.18 1.66 0.15
Hearth rate (beats/min) 69 12 75 18 71 16 81 11
Office SBP (mmHg) 112.6 5.1 118.4 5.3 153.4 9.7*** 154.4 9.6***
Office DBP (mmHg) 66.8 6.1 69.1 8.1 89.4 9.4*** 89.7 8.4***
24-h SBP (mmHg) – – 143.6 8.2 144.4 9.9
24-h DBP (mmHg) – – 80.6 6.1 79.4 9.4
Blood glucose (mmol/l) 4.13 0.33 4.51 0.21 4.08 0.22 4.68 0.38*
Cholesterol (mmol/l) 4.16 0.22 4.52 0.26* 4.23 0.37 4.55 0.24*
Triglycerides (mmol/l) 1.17 0.18 1.44 0.41 1.21 0.22 1.53 0.33*
Creatinine (μmol/l) 67 5 82 8 76 8 81 7
Data presented as mean standard deviation. SBP, systolic blood pressure; DBP, diastolic blood
pressure. ***P < 0.001 versus controls; *P < 0.05 versus BMI < 25.
20
3.2. Clinical characteristics of subjects in the genetic study of renin-angiotensin system
Thirty-five adolescents with EH (mean age 14.4 2.7 years, male/female 30/5), and 70
patients with ESRD (20 pediatric, mean age 14.9 ± 3.1 years, male/female 9/11 and 50 adult,
mean age 48.7 ± 18.7 years, male/female 23/27) were genotyped for RAS polymorphisms in
the genetic study. One hundred and thirty healthy randomly selected normotensive blood
donors from the blood bank of our university (mean age 34.9 8.1 years, male/female 66/64,
BP 117.9 ± 8.7/78.7 ± 8.5 mmHg) and 20 healthy children with normal BP from a school
screening program (mean age 13.2 1.2 years, male/female 10/10, BP 109 ± 6.5/71 ± 5.9
mmHg) were also studied as controls to determine the frequency of gene polymorphisms
within the population. No differences were observed regarding the genotype and allele
distributions between the pediatric and adult control groups, therefore the values have been
collected in one group as controls (n = 150). In the control group the genotype distribution is
consistent with Hardy-Weinberg equilibrium.
The diagnosis of hypertension was set up according to the same definition and clinical
evaluation protocol which we used in the biochemical study. All the hypertensive patients in
the genetic study had a BMI below 30 kg/m2, and normal serum cholesterol (< 5.2 mmol/l)
and triglyceride (< 1.7 mmol/l) levels.
In patients with ESRD, the hereditary nephrological diseases, myeloma multiplex, and
malignant disorders were excluded. The distribution of the original nephrological diagnoses in
the adult ESRD group was as follows: chronic pyelonephritis 31, IgA nephropathy 6, focal
segmental glomerulosclerosis (FSGS) 5, membranoproliferative glomerulonephritis 6, rapidly
progressive glomerulonephritis 2. In the pediatric ESRD group the distribution was chronic
pyelonephritis with reflux nephropathy 12, tubulointerstitial nephritis 2, membrano-
proliferative glomerulonephritis 3, FSGS 2, and rapidly progressive glomerulonephritis 1.
ESRD patients were on routine bicarbonate hemodialysis (all patients were treated three times
per week, mean treatment time was 4.08 ± 0.59 h; Gambro dialysis machines were used). In
ESRD patients the mean concentrations of total serum cholesterol (5.5 ± 1.2 mmol/l) and
triglyceride (2.5 ± 0.7 mmol/l) were slightly above the laboratory references (normal
cholesterol < 5.2 and triglyceride < 1.7 mmol/l).
21
Demographic data, physical characteristics and some related metabolic parameters of the
hypertensive and ESRD patients in the genetic study are presented in Table 3.
Table 3. Demographic data, physical characteristics and some metabolic parameters
of patient groups in the genetic study of renin-angiotensin system
ESRD patients (n = 70)
Hypertensive
patients (n = 35) Pediatric
(n = 20) Adult
(n = 51)
Age (years) 14.4 2.7 14.9 ± 3.1 48.7 ± 18.7
Sex (male/female) 30/5 9/11 23/27
SBP (mmHg) 135.4 7.4 149.1 ± 24 139 ± 14
DBP (mmHg) 72.4 7.7 96.9 ± 12 91 ± 13
BMI (kg/m2) 25.3 2.7 –
Cholesterol (mmol/l) 3.9 0.5 5.5 ± 1.2*
Triglyceride (mmol/l) 1.1 0.4 2.5 ± 0.7*
Creatinine (μmol/l) 80 15.4 785 ± 129
Data presented as mean standard deviation. ESRD, end stage renal disease; SBP, systolic blood
pressure; DBP, diastolic blood pressure; BMI, body mass index. SBP and DBP values are
expressed as 24-h mean in hypertensive patients and single measurement mean, before
hemodialysis in ESRD patients. Serum cholesterol, triglyceride and creatinine were measured
before hemodialysis. *mild increase compared to the laboratory reference values (normal
cholesterol < 5.2 and triglyceride < 1.7 mmol/l).
The studies were previously approved by the Ethical Committee of the University of Szeged.
Informed consent was obtained from the participants or from their parents.
22
4. METHODS
4.1. Biochemical methods
Nitric oxide end-products in plasma (nitrite + nitrate, NOx)
NO reduction is considered the most important factor in the development of endothelial
dysfunction. NO is oxidized in the blood and tissues to form nitrite and nitrate. These end-
products are generally accepted markers of endogenous NO production. The plasma level of
the sum of nitrite and nitrate (NOx) is frequently used to assess NO bioavailability in vivo,
which reflects the degree of endothelial dysfunction in humans [40, 41].
Nitrite and nitrate were determined simultaneously by an anion-exchange high-
performance liquid chromatography (HPLC) method [64]. The Pharmacia LKB HPLC system
was used with a Variable Wavelength Ultraviolet Detector (at 210 nm). The within-day (intra-
assay) coefficient of variation (CV) for nitrate standards in pooled plasma ranged from 4.5%
for 25 mol/L to 2.5% for 50 mol/L (n 10). The day-to-day (inter-assay) variability was
5.2% for 25 mol/L and 3.5% for 50 mol/L (n 20).
Plasma lipid peroxides as malondialdehyde equivalents
LPO is the oxidative degeneration of lipids caused by ROS. In the course of this process a
number of by-products are generated, such as malondialdehyde (MDA), and they are
commonly used as oxidative stress markers [23, 24].
Plasma lipid peroxides were quantified as MDA-thiobarbituric acid adducts by the
HPLC method [65], using a 3.9 × 300mm Bondapak C18 (10 m) (Waters-Millipore Corp.,
Milford, Massachusetts, USA) reverse-phase HPLC column and a Variable Wavelength
Ultraviolet Detector (at 532 nm). The reproducibility of the results was evaluated by the
analyses of commercial quality-control serum. Replicate daily analyses yielded a CV of
12.1% at 1.19 mol/L (n 18), while the intra-assay CVs were below 8% (n 10).
Plasma free thiol groups
Thiols, such as glutathione, cysteine and homocysteine, are organosulfur compounds that
contain a carbon-bonded thiol (sulfhydryl) group. Among all the antioxidants that are
23
available in the body, intracellular and extracellular thiols constitute the major portion of the
total body antioxidants and they play a significant role in the defense against ROS. In the
plasma both free and protein-bound thiols are presented. The plasma level of free thiol groups
is a useful marker of oxidative stress; they correlate inversely with each other [66].
Plasma free thiol groups were assayed with 5,5-dithiobis (2-nitrobenzoic acid) at
412 nm a molar extinction coefficient of 13 600 was used [67]. The values of both the inter-
and the intra-assay precision were similar to those reported by other authors, with CVs of
8.9% and 6.7% in the presence of 300 mol/L free thiol, as a GSH standard [67].
Biochemical analysis of the glutathione redox system
Glutathione tripeptide in its reduced state (GSH) is present in millimolar intracellular
concentrations, and provides antioxidant protection in all the cells of the body. In the presence
of oxidative stress GSH is converted to oxidized glutathione (glutathione disulfide, GSSG),
which is reduced back to GSH by the enzyme glutathione reductase using NADPH as an
electron donor. The GSH concentration in the plasma has previously been used as a marker of
oxidative stress in hypertensive patients [68].
Red blood cells (RBC), contain millimolar concentrations of GSH, and circulate in the
direct vicinity of the endothelial surface. Their membranes are permeable to oxidants and
therefore can provide antioxidant protection to their surroundings [69]. Inter-related enzyme
systems in the RBC function to achieve the efficient recycling of GSSG to GSH and to
provide the reducing equivalent, NADPH. Hence, GSH concentration, and the redox ratio
GSSG/GSH of the RBC can serve as a reliable parameter of the oxidative imbalance in
patients with endothelial dysfunction (GSH concentration decreases, while GSSG/GSH ratio
increases).
Highly sensitive and specific separate determinations of GSSG and GSH + GSSG
concentrations were carried out by a previously published method [70]. This is a combination
of standard methods [71] used after validation for accurate determination, especially of GSSG
values in the presence of much higher concentrations of GSH (more than 50 times), in the
presence of hemoglobin (Hb). The intra-assay and inter-assay variabilities of the assay, using
GSSG standard at 50 nmol/l, resulted in CVs of 3.5% (n = 10) and 5.8% (n = 20), respectively
[70].
24
The „GSH stability test” [72] was used to measure the recycling capacity of the RBC after an
in vitro oxidative stress. Acetylphenylhydrazine (APH) (0.33 mmol/l) was added to the whole
blood sample, together with sufficient glucose. Following incubation at 37 C for 60 min with
APH, RBC deficient in recycling, but not the normal RBC, suffer a marked fall in GSH level
[72]. This method of calibrated oxidative challenge was established by Beutler in order to
recognize patients with deficient glucose-6-phosphate-dehydrogenase [72] and was proved to
be a sensitive biochemical marker of the oxidative susceptibility of the RBC both in
population studies and during infection [73, 74].
The proportions of carbon monoxide hemoglobin and methemoglobin, and the total
concentration of hemoglobin in the whole blood were measured with a Hemoximeter
(Radiometer, Copenhagen, Denmark) within 15 min after venipuncture.
4.2. Determination of renin-angiotensin system gene polymorphisms
Genomic DNA was isolated from peripheral blood leukocytes by a standard
phenol/chloroform method [75].
I/D polymorphism of the angiotensin-converting enzyme (ACE) gene
The I/D polymorphism in intron 16 of the ACE gene was determined according to the
previously published method of Chiu and McCarthy [76] using polymerase chain reaction
(PCR). A forward primer (5’-CTGGAGACCACTCCCATCCTTTCT-3’), a reverse primer
(5’-TCGAGACCATCCCGGCTAAAAC-3’), and an insertion-specific primer (5’-GAT
GTGGCCATCACATTCGTCAGAT-3’) were used for the PCR. Amplification was carried
out in a 25 l reaction mixture containing 250 ng genomic DNA, 2 mM MgCl2, 50 mM KCl,
10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 5% dimethylsulfoxide, 0.2 mM of each dNTP,
25 pmol of each primer and 0.25 U Taq DNA polymerase. The amplification involved the
following steps: 5 min initial denaturation at 94 ºC, followed by 30 cycles of 1 min
denaturation at 94 ºC, 1 min annealing at 64 ºC, 1 min extension at 72 ºC, and then 10 min
final extension at 72 ºC. The samples were visualized after electrophoresis on a 2% agarose
gel with ethidium bromide staining. The DD genotypes were re-typed with the insertion
specific primer and the consensus (forward) primer.
25
M235T polymorphism of the angiotensinogen gene (AGT)
The M235T polymorphism in exon 2 of the AGT gene was detected by a single-step Light
Cycler technology published by Malin et al. [77] that uses a rapid PCR amplification followed
by the analysis of the melting behaviour of fluorophore-labeled hybridization probes. As a
reaction buffer in the PCR, the Light Cycler DNA Master Hybridization Probes 10× buffer
(Roche Molecular Biochemicals) with a final Mg2+ concentration of 4 mmol/L was used. PCR
was performed in a reaction volume of 20 l with 0.3 mol/L of each primer (5’-
CTCTATCTGGGAGCCTTG-3’ and 5’-GTTTGCCTTACCTTGGAA-3’) and 0.2 mol/ L of
the anchor and detection probes. The detection probe was labelled at the 3’ end with
fluorescein (5’-CCCTGAGTGGAGCCAGTG-X); the anchor probe was labelled with
LightCycler Red 640 at its 5’ end and modified at the 3’ end by phosphorylation to block
extension (5’-LC Red 640-GACAGCACCCTGGCTTTCAACAC-P). The PCR was
performed in a LightCycler instrument and included initial denaturation at 94 C for 45 sec,
followed by 50 cycles of denaturation (94 C for 0 sec, with a temperature transition rate of
20 C/sec), annealing (57 C for 5 sec, 20 C/sec), and single extension (72 C for 20 sec, 3
C/sec). After the amplification, the melting curve was recorded by cooling the reaction
mixture to 50 C for 3 min, and then by slowly raising the temperature to 85 C at 0.2 C/sec.
The fluorescence signal (F) was continuously monitored during the temperature ramp and
then plotted against the temperature (T) to obtain melting curves for the samples (F vs. T).
The melting curves were subsequently converted to derivative melting curves [–(dF/dT) vs.
T]. The melting peak of the samples homozygous for the M allele was at 63 C, whereas in
samples homozygous for the T allele, the melting peak was at 53 C. The heterozygous
samples contained both M and T alleles and produced both peaks.
A1166C polymorphism of the angiotensin II type 1 receptor (AT1R) gene
Determination of the A1166C polymorphism of the AT1 gene was also carried out with
LightCycler technology according to the M235T polymorphism detection (described above)
which was optimized for the analysis of A1166C polymorphism by us.
The same PCR reaction buffer with a final Mg2+ concentration of 3 mmol/L and 20 l
reaction volume with 0.4 mol/L of each primer (5’-ATCCACCAAGAAGCCT-3’ and 5’-
AAAGTCGGTTCAGTCCA-3’) and 0.2 mol/L of hybridization probes were used for the
26
PCR. The detection probe was labelled at the 3’ end with fluorescein (5’-
AGGAGCAAGAGAACATTCCTCTGCA-X) and the anchor probe with LightCycler Red
640 at its 5’ end and modified at the 3’ end by phosphorylation (5’-LC Red 640-
ACTTCACTACCAAAGTAGCCTTAGC-P). The amplification included an initial
denaturation at 94 C for 45 sec, followed by 50 cycles of denaturation at 94 C for 5 sec,
annealing at 57 C for 15 sec and single extension at 72 C for 25 sec. After the PCR the
melting curves were recorded by cooling the reaction mixture to 40 C for 2 min and then by
slowly raising the temperature to 85 C at 0.2 C/sec. The melting peaks of the AA and CC
genotypes were 61 C and 67 C.
4.3. Statistical analysis
Clinical data on the patients are reported as means ± standard deviations. The results of
biochemical analyses are shown in the figures as means ± standard errors. Statistical analyses
included both parametric (variance analysis, Tukey test and Students t test) and non-
parametric tests (Wilcoxon rank test, chi-square test) as appropriate. When the extent of
variance between pairs of groups differed significantly (P < 0.05 in the F test), we used the
Welch test (d probe) instead of the t test to compare the mean values. Correlations between
parameters were characterized by calculation of the linear regression and correlation
coefficients. The distribution of genotypes was expressed as percentage frequency and odds
ratio (OR) values were also calculated. Deviation from Hardy-Weinberg equilibrium was
assessed by chi-square test with Yates correction. P less than 0.05 was considered significant
for all statistical tests.
27
5. RESULTS (according to the main questions of the studies)
Question 1. Is there any relationship between BP and BMI?
Result 1. In the hypertensive patients, the BMI displayed significant positive correlations with
both the SBP and the DBP (r = 0.581 and r = 0.542, respectively; n = 52, P < 0.001).
Question 2. Does NO production, which is characterized by plasma concentration of its
end-products (NOx), decrease in juvenile EH? Does it depend on BMI?
Result 2. High decreases in plasma NOx concentrations were only seen in hypertensive
patients with a normal body mass (BMI < 25 kg/m2) compared to the control subjects. In
overweight groups (BMI > 25 kg/m2) the NOx levels were somewhat, but not significantly,
lower, irrespectively of hypertension (Figure 2).
Figure 2. Plasma concentration of nitric oxide metabolites (nitrite + nitrate, NOx) in controls and
hypertensive patients, related to their body mass index (BMI), means ± standard errors. **P < 0.01
versus BMI-matched controls.
28
The plasma NOx concentration did not show significant correlations neither with BMI nor the
investigated parameters of oxidative stress.
Question 3. Can we observe an increased oxidative stress state in juvenile EH? Is there any
correlation between oxidative stress and BP? Does the oxidative stress correlate with BMI
in normotensive and hypertensive adolescents?
Result 3. The MDA concentrations were significantly higher only in case of overweight
hypertensive patients (BMI > 25 kg/m2) as compared with the levels of the BMI-matched
control subjects (Figure 3).
Figure 3. Plasma concentration of end-products of lipid peroxides (malondialdehyde, MDA) in
controls and hypertensive patients, related to their body mass index (BMI), means ± standard
errors. **P < 0.01 versus BMI-matched controls.
29
The concentrations of free thiols displayed a marked decrease in overweight normotensive
subjects, and a slight reduction in overweight hypertensive patients (Table 4), as a sensitive
sign of a moderate pro-oxidant state related to overweight state even without hypertension.
Table 4. Concentration of some metabolic parameters in control subjects and
hypertensive patients, related to their body mass index (BMI, kg/m2)
Controls (n = 48)
Hypertensive patients (n = 52)
BMI < 25 (n = 26)
BMI > 25 (n = 22)
BMI < 25 (n = 28)
BMI > 25 (n = 24)
Whole blood values
Total hemoglobin (mmol/l) 9.2 0.9 9.3 0.7 9.9 0.8* 9.8 0.7*
Carboxyhemoglobin (μmol/l) 144 43 165 57 158 63 179 78
Methemoglobin (μmol/l) 82 16 82 13 86 25 90 23
Oxidized glutathione (GSSG, nmol/g Hb) 9.5 2 11.4 3 12.5 3* 14.1 3
Plasma values
Free thiols (μmol/l) 215 35 181 24 209 38 199 30
Data presented as mean standard deviation. Hb, hemoglobin. *P < 0.05 versus BMI-matched
controls; P < 0.05, P < 0.001 versus BMI < 25.
The GSH levels were considerably decreased in hypertensive patients, irrespective of their
BMI (Figure 4a). After in vitro oxidant insult, there was a gradual decrease in the proportion
of residual GSH with increasing BMI, as a sign of the potential effects of certain metabolic
factors on the GSH stability. Nevertheless, a further significant fall in the residual GSH was
observed in the hypertensive non-obese patient groups (BMI < 30) as compared with the
BMI-matched control groups (Figure 4b).
30
The significant depletion of erythrocyte GSH in hypertensive patients with somewhat
increasing values of GSSG according to BMI (Table 4) resulted in a highly elevated redox
ratio GSSG/GSH in patients with hypertension as compared with the BMI-matched controls
(Figure 5a). However, there was not a significant direct relationship between the BP and
GSSG/GSH.
When the concentrations of LPO end-products (MDA) were related to those of NOx, a
highly significant increase was seen in hypertensive patients, with or without overweight
condition (Figure 5b). In addition, the ratios MDA/NOx proved to correlate significantly with
both the SBP and the DBP in the overall patient population (r = 0.525 and r = 0.492,
respectively; n = 100, P < 0.001). Age was not a significant covariate in this correlation. None
of the oxidative stress parameters correlated significantly with BMI.
Figure 4. Concentrations of reduced glutathione (GSH) (a) and the proportion of GSH remaining
after in vitro oxidative stress with acetylphenylhydrazine (b) in the red blood cells of controls and
hypertensive patients, related to their body mass index (BMI), means ± standard errors. *P < 0.05, **P < 0.01, ***P < 0.001 versus BMI-matched controls.
31
Figure 5. Ratios of oxidized/reduced glutathione (GSSG/GSH) (a) and lipid peroxides/nitric oxide
metabolites (MDA/NOx) (b) as parameters of oxidative stress in controls and hypertensive
patients, related to their body mass index (BMI), means ± standard errors. *P < 0.05, **P < 0.01, ***P < 0.001 versus BMI-matched controls.
32
Question 4. May the polymorphisms of the RAS genes have any influence on the develop-
ment of juvenile EH?
Result 4. Genotype and allele frequencies for the polymorphisms of the RAS genes in
hypertensive patients are listed in Table 5 and Table 6. A significant increase in the
frequency of the MT genotype of the AGT gene was observed in juvenile EH (P < 0.02), the
OR was 2.9, respectively. The frequencies of the other AGT genotypes, and also frequencies
of all ACE and AT1R genotypes did not differ significantly in the hypertensive and control
group, only a weak non significant increase in the incidence of the II genotype of ACE gene
was seen. Furthermore, the same allele frequencies of the M235T polymorphism of AGT
gene, I/D polymorphism of the ACE gene, and A1166C polymorphism of the AT1R gene
were found in the hypertensive as well as the control group.
Table 5. Genotype frequencies (%) for the M235T polymorphism of the
angiotensinogen (AGT) gene, I/D polymorphism of the angiotensin converting enzyme
(ACE) gene, and A1166C polymorphism of the angiotensin II type 1 receptor (AT1R)
gene in hypertensive patients, and controls (OR, odds ratio)
Hypertensive patients (n = 35)
Gene polymorphism
Genotype
n (%) OR
Controls (n = 150)
n (%)
MM 4 (12) 0.4 37 (25)
MT 26 (74)**
2.9 75 (50) AGT M235T
TT 5 (14) 0.5 38 (25)
II 12 (34) 1.8 34 (23)
ID 16 (46) 0.7 83 (55) ACE I/D
DD 7 (20) 0.9 33 (22)
AA 13 (37) 0.6 78 (51)
AC 20 (57) 1.6 67 (45) AT1R A1166C
CC 2 (6) 1.8 5 (4)
**P < 0.02
33
Table 6. Allele frequencies for the M235T polymorphism of the angiotensinogen
(AGT) gene, I/D polymorphism of the angiotensin converting enzyme (ACE) gene,
and A1166C polymorphism of the angiotensin II type 1 receptor (AT1R) gene in
hypertensive and end stage renal disease (ESRD) patients, and controls
ESRD Gene polymorphism
Alleles Hypertensive patients (n = 35)
Pediatric (n = 20)
Adult (n = 50)
Controls (n = 150)
M 0.49 0.50 0.55 0.50 AGT M235T
T 0.51 0.50 0.45 0.50
I 0.56 0.35 0.47 0.50 ACE I/D
D 0.44 0.65* 0.53 0.50
A 0.69 0.78 0.72 0.75 AT1R A1166C
C 0.31 0.22 0.28 0.25
*P < 0.05
Question 5. Is there any role of the RAS gene polymorphisms in the development of ESRD?
Result 5. There was a significant increase in the frequency of the MT genotype of the AGT
gene in the group of pediatric ESRD patients (P < 0.02), the OR was 4.0, respectively. The
DD genotype of the ACE gene was over-represented in pediatric ESRD patients compared
with that of the control group (pediatric 45% versus controls 22%; P < 0.05). The OR was
2.9. In addition, a non-significant increase of the DD genotype frequency was found in the
adult ESRD group. The similar genotype distributions of the AT1R gene polymorphism were
observed in the ESRD and control groups. The slightly higher CC genotype frequency, which
we have found in adult ESRD patients compared to that of the control group, was not
significant (Table 7). Allele frequencies of the M235T polymorphism did not differ in
pediatric and adult patients with ESRD compared with the controls. Similarly, there was no
significant difference between the ESRD groups and the control group regarding the allele
frequencies of AT1R gene polymorphism. The D allele of the ACE gene was significantly
more frequent in the pediatric ESRD patients compared with the control subjects (P < 0.05)
(Table 6).
34
Table 7. Genotype frequencies (%) for the M235T polymorphism of the
angiotensinogen (AGT) gene, I/D polymorphism of the angiotensin converting enzyme
(ACE) gene, and A1166C polymorphism of the angiotensin II type 1 receptor (AT1R)
gene in end stage renal disease (ESRD) patients, and controls (OR, odds ratio)
ESRD
Pediatric (n = 20)
Adult (n = 50)
Gene polymorphism
Genotype
n (%) OR n (%) OR
Controls (n = 150)
n (%)
MM 2 (10) 0.4 17 (34) 1.6 37 (25)
MT 16 (80)** 4.0 21 (42) 0.7 75 (50) AGT M235T
TT 2 (10) 0.3 12 (24) 0.9 38 (25)
II 3 (15) 0.6 11 (22) 1.0 34 (23)
ID 8 (40) 0.5 25 (50) 0.8 83 (55) ACE I/D
DD 9 (45)* 2.9 14 (28) 1.4 33 (22)
AA 11 (55) 1.1 26 (52) 1.0 78 (51)
AC 9 (45) 1.0 20 (40) 0.8 67 (45) AT1R A1166C
CC – – 4 (8) 2.5 5 (4)
*P < 0.05, **P < 0.02
35
6. DISCUSSION
6.1. Oxidative stress and endothelial dysfunction in juvenile essential hypertension and
obesity
The BMI is a clinical indicator of overall body fat [78]. We used it as a definition of the mass
status based on height and weight, because these can be obtained with reasonable precision.
Comparisons of various weight-for-height indices for both adults and children have led to the
selection of the BMI as the most desirable parameter [79]. The BMI cut-off points in the
adolescent age group do not differ significantly from those of the adults; a value above 25
indicates overweight status, and one above 30 indicates obesity [79].
In our hypertensive patients, both the systolic and the diastolic BP correlated
significantly positively with the BMI, indicating that the weight status is a confounding factor
in the assessments of the potential relationship between oxidative stress and hypertension.
The positive correlations between the BP values and weight, BMI and height in childhood
were demonstrated by several studies in large populations of children, among others in
Hungarian adolescents [80].
Impairment of NO bioavailability, which is a major component of endothelial
dysfunction, plays an important role in the mechanism of hypertension [39]. Determination of
stable end-products of NO, nitrite and nitrate (NOx) in plasma is often used as an index of
systemic NO formation [40, 41], which reflects not only NO production and NOS activity, but
also NO quenching by ROS, therefore may allude to an oxidative stress state. Plasma NOx
level significantly decreased in hypertensive patients with normal BMI. In overweight
children, irrespectively of hypertension, a non-significant decrease in plasma NOx was
observed. In a previous study from our group a marked decrease was seen in plasma NOx in
non-obese and also in obese hypertensive patients [44]. These data suggest that endothelial
dysfunction, characterized by decreased plasma NOx may be a more important mechanism in
EH of lean patients than in OH, but the presence of endothelial dysfunction is detectable in
obesity and OH. Moreover, decrease in plasma NOx may be an indirect sign for the increased
oxidative stress in hypertensive and obese subjects.
36
In the present study, the concerted action of the mass status and hypertension was measured in
terms of the different biochemical parameters of oxidative stress. However, the results from
hypertensive patients, compared with BMI-matched control subjects, permit identification of
the differences resulting exclusively from the hypertensive state. As a sensitive sign of the
compromised antioxidant status the plasma level of free thiols was reduced in overweight
patients, even in case of those without hypertension. In contrast, a significant increase in the
end-products of lipid peroxides, in MDA concentrations, was observed only in cases
involving both hypertension and overweight state. However, the ratio MDA/NOx, as a
measure of the increase in LPO relative to NO, proved to be a useful marker of the link
between BP and oxidative stress, presenting a highly significant increase in hypertensive
patients irrespective of their BMI. The significant correlation between MDA/NOx and the
systolic and diastolic BP from a clinical point of view makes this parameter important.
In adult patients with uncontrolled EH, the levels of MDA were high, whereas those of
NOx were low [81]. When the ratio of lipid peroxides/NO was calculated, a far more
significant rise in the concentration of lipid peroxides as compared to NOx levels was noted
[81]. Our results in young hypertensive patients with a different mass status are in accordance
with the conclusion that the ratio MDA/NOx is a BMI-independent sensitive parameter of the
oxidative stress in childhood and adolescent hypertension. Additionally, the ratio was
normalized following the control of hypertension in the study of Kumar and Das [81]. Thus,
this ratio may be used as a relevant target for future clinical trials aimed at controlling the
effects of antioxidant or antihypertensive treatment in patients with juvenile EH.
However, an oxidative stress status irrespective of BMI was also consistently
manifested by a depleted GSH level and a pronounced GSH lability after APH loading in
hypertensive adolescents. Another marker of oxidative stress, the redox ratio GSSG/GSH in
RBC, also increased significantly in hypertensive patients, independent of their BMI.
Although GSH depletion was the predominant cause, a higher GSSG in overweight patients
had some influence as well. This could be the explanation for the absence of direct correlation
between the BP values and GSSG/GSH.
These findings prove the presence of increased oxidative stress in childhood EH. In
addition, a significantly positive correlation was observed between oxidative stress,
characterized by MDA/NOx ratio and BP. The extent of oxidative stress was not correlated
37
directly to BMI, but obese hypertensive patients exhibited a slightly more pronounced
alterations in oxidative stress parameters than hypertensive patients with normal BMI. A
compromised antioxidant status as the sign of increased oxidative stress was observed in
obese control subjects. Obesity per se is a pro-oxidant state, and may aggravate the oxidative
stress state if it is associated with hypertension.
The present study does not give an answer to the question of whether the oxidative
stress is the cause or consequence of hypertension. GSH depletion resulted in a perturbation
of the NO system and caused severe hypertension in normal animals [82]. The administration
of antioxidant vitamins ameliorated hypertension and improved the urinary nitrate-nitrite
excretion, supporting the notion that oxidative stress is involved in the pathogenesis of this
experimental hypertension [82]. On the other hand, thiol supplementation with GSH, given by
intravenous infusion, selectively improved a human endothelial dysfunction by enhancing NO
activity [83]. Moreover, a GSH infusion caused a reduction of BP in adult hypertensive
patients [84].
In conclusion, an imbalance between the available NO and LPO end-products, with a
simultaneous alteration in the glutathione redox system of the RBC, was present in young
hypertensive patients, irrespective of their weight status. The population studied was
relatively small and rather heterogenous with regard to age. Thus, further studies are
warranted to clarify the relationship between the hormonal, SNS activity and the redox status
during the age of puberty.
6.2. Gene polymorphisms of the renin-angiotensin system in juvenile essential
hypertension and uremia
Genetic association studies based on the comparison of genotype and allele distribution in
cases and controls are considered a useful approach in studying the role of candidate genes in
the development and progression of multifactorial diseases, such as hypertension, and other
cardiovascular or chronic renal diseases. Among the candidate genes, AGT, ACE and AT1R
genes of the RAS seem to be particularly biologically and clinically relevant to cardiovascular
and renal diseases [85].
38
Evidence has been suggesting that RAS and Ang II have a central role in the pathogenesis of
cardiovascular and chronic renal diseases, such as LVH and fibrosis, vascular media
hypertrophy or neointima formation, and structural alterations of the heart and the kidney [60,
86]. Therefore, genetic variants of the RAS, which have influence on the production or action
of Ang II, may have a pathogenetic role in these disorders.
Large numbers of association studies have been published about the possible role of
polymorphisms of the RAS genes in cardiovascular and renal diseases. The results are often
inconclusive and sometimes different studies may even be conflicting. The interpretation of
data is dependent on several aspects, for example the sample size, clinical and genetic
homogenity of the study population, definition and relevancy of the outcome, the role of
acquired and environmental factors [87]. All of these factors may have a significant influence
on the results and conclusions. The differences in the prevalence of a polymorphism between
various ethnic groups may also be an influential factor.
The distribution of the ACE gene polymorphisms in the Caucasian population is similar
in different European countries; with incidences of II, ID, and DD genotypes in Germany [88]
of 26%, 50%, and 27%, respectively; in France [89] of 22%, 51%, and 27%; in Sweden [90]
of 23%, 50%, and 27%. In the present study we accumulated data for the Hungarian
population (23%, 55%, 22%) that were in accordance with these studies. In Japanese [61] and
South Asian [91] control groups the incidence of II genotype was higher (41% and 39.8%,
respectively) than in the European population. In the United States it was lower, but the
incidence of DD genotype was higher than in the European countries [92].
In a large study of hypertensive siblings, a strong association was demonstrated between
M235T polymorphism of the AGT gene and hypertension, which was later confirmed in
patients with no family history of hypertension [93, 94]. In several other studies, the role of
M235T polymorphism was demonstrated in cardiovascular disorders and hypertension. These
reported that the TT genotype of AGT was associated with an increased risk of hypertension
and coronary heart disease [95, 96]. Theoretically, this polymorphism accounts for higher
angiotensinogen levels, which could translate into higher Ang II levels and may lead to
pathological changes in the cardiovascular tissues. This concept was confirmed by a case-
control study of Winkelmann et al., in which a higher plasma angiotensinogen levels were
linked to the number of T235 alleles and to elevated DBP. Furthermore, they observed a weak
39
association between the M235T variant and coronary artery disease and myocardial infarction
[48]. In contrast, Caulfield et al. failed to show an association between AGT gene
polymorphism and hypertension [97].
The results of Gumprecht et al. [98] suggest that the AGT gene M235T polymorphism
contributes to the increased risk for the development of CRF. The T235 allele was transmitted
more frequently to patients with CRF caused by interstitial nephritis. In type 2 diabetes an
association of AGT gene polymorphism with renal dysfunction and coronary heart disease
was also found [99]. T allele and TT genotype showed a significant association with
albuminuria in Chinese patients with type 2 diabetes and Japanese children with IgA
nephropathy [100, 101]. Others were not able to confirm the role of this polymorphism in the
development or progression of chronic renal diseases [102–104]. A fairly conclusive meta-
analysis of the association of AGT gene M235T polymorphism with ESRD was published by
Zhou et al. based on sixteen literatures. T allele and TT genotype were associated with ESRD
susceptibility in Caucasians, which were not observed in overall populations, Asians and
Africans [105].
We did not demonstrate an increased frequency of the TT genotype in juvenile EH and
ESRD as expected from previous studies, but there was a significant increase in MT genotype
in the groups of EH and pediatric ESRD. It is conceivable, that the small number of patients
in these groups did not allow detection of an increased incidence of the TT genotype.
A study by Zee et al. [106] is the only report that demonstrates a positive association
between the ACE gene and hypertension. A higher frequency of I allele and lower frequency
of the DD genotype were found in adult hypertensive patients with a family history of
hypertension compared with normotensive subjects. Similarly, a weak, non significant
increase in the incidence of the II genotype, but normal frequencies of the ID and DD
genotypes were found in our adolescent essential hypertensive patients. Schunkert et al. [107]
and Iwai et al. [108] reported a positive association between DD genotype and LVH, but did
not identify a relationship between hypertension and ACE gene polymorphism. The relevance
of ACE I/D polymorphism in cardiovascular diseases was also showed by the ECTIM study
[109]. Subjects with DD genotype had an increased risk of myocardial infarction. Data from
Butler et al. suggest that DD genotype is associated with arterial dysfunction limited to NO
pathway [110].
40
We found an increased prevalence of the D allele and DD genotype in the pediatric ESRD
population, suggesting that certain pathogenetic mechanisms associated with the D allele
result in a faster progression of ESRD in pediatric kidney disorders. These results are in
accordance with those of the multicenter study of Hohenfellner et al. [111]. They found that
the ACE DD genotype is a significant risk factor for children with congenital renal
malformations associated with progressive CRF. Although hereditary renal diseases were
excluded from our study, reflux nephropathies associated with pyelonephritis were included.
The role of D allele and DD genotype in the progression of CRF has been demonstrated
in a large number of nephropathies of different origin. Ozen et al. found that the DD genotype
had a significant impact on renal scar formation in reflux nephropathy [112]. This association
was also confirmed in our study of patients with vesicoureteric reflux [113]. In a group of
children with FSGS homozygotes for the I allele were less likely to have progressive renal
disease than in case of patients with other genotypes (ID and DD), so the II genotype had a
protective, while the D allele had a detrimental effect on the outcome in this study [114].
Studies in IgA nephropathy showed that ACE genotype is a risk factor for the worsening of
nephropathy clinical course; patients with II genotype have more favorable prognosis than
those with ID and DD [115, 116]. The role of the D allele in progression of non-diabetic CRF
patients was confirmed by the study of Samuelson et al. [117]. The progression of CRF to
ESRD was faster in patients with autosomal dominant polycystic kidney disease (ADPKD)
who had DD genotype, compared with other genotypes [118]. The II genotype is protective
against the development and progression of diabetic nephropathy and is associated with a
slower progression of non-diabetic proteinuric kidney disease published in a review by
Ruggenenti et al. [87]. Our adult ESRD group showed a slight increase in DD genotype
frequency (OR = 1.4). The difference between pediatric and adult ESRD patients in DD
genotype frequency may be an explanation for the faster progression in the decline of renal
function. In contrast to our findings and studies cited above, others failed to demonstrate a
relationship between ACE I/D polymorphism and the progression of various chronic renal
diseases [102, 103, 119].
Most of the known actions of Ang II are mediated by the AT1R [53]. The functional
significance of the A1166C non-coding polymorphism of AT1R gene is uncertain, and its
influence on Ang II responsiveness is not confirmed. Nevertheless, studies have revealed an
41
association between this polymorphism and hypertension [120] and aortic stiffness in
hypertensive patients [121]. A significant association of the C1166 allele to essential
hypertension was confirmed by other studies [122, 123]. Moreover, a synergistic interaction
between the AT1R and ACE gene polymorphism on the risk of myocardial infarction was
demonstrated [124], but in the ECTIM study this genetic variant of AT1R did not affect the
risk of myocardial infarction [125]. In addition, other studies in Caucasian populations or in
Japanese persons were not able to demonstrate an obvious link between A1166C
polymorphism and hypertension [53]. An increased response to Ang II was observed in
isolated arteries from patients with CC genotype. This association suggests that the A1166C
polymorphism may alter Ang II responsiveness indirectly (mostly in linkage with a functional
mutation), which may explain the relation between this polymorphism and cardiovascular
abnormalities [126].
About the role of A1166C polymorphism of AT1R gene in chronic renal diseases
conflicting results were published, an association was found between this polymorphism and
renal function in diabetic nephropathy [99], but was not in IgA nephropathy [103] and
ADPKD [104].
We found a non-significant increase of the CC genotype in the groups of juvenile EH
and adult ESRD patients (the ORs were 1.8 and 2.5, respectively, P > 0.05). The frequency of
the AC genotype in hypertensive patients was higher (57%) compared with that of the control
group (45%), but it was not significant either.
In summary, there is no difference in the ACE genotype distribution between Hungarian
and other Caucasian populations. We conclude that the DD genotype of ACE was more
frequent in pediatric ESRD. This genotype, which is associated with higher circulating and
tissue ACE levels, could be a genetic risk factor for renal parenchymal destruction, renal
scarring, and the development of ESRD in children, independent of the original renal disease.
Furthermore, a significant increase was observed in the occurrence of AGT MT genotype in
the groups of hypertensive and pediatric ESRD. The role of the AT1R gene polymorphism in
juvenile EH and ESRD needs to be further investigated.
42
7. SUMMARY OF OUR FINDINGS AND CONCLUSIONS (according to the main
questions of the studies)
Conclusion 1. Obesity is directly related to blood pressure (BP) and hypertension. In
accordance to this statement, the body mass index (BMI) displayed significant positive
correlations with both the systolic and the diastolic BP in our hypertensive patients.
Conclusion 2. A significant decrease in plasma concentration of nitric oxide end-products
(NOx) was observed in hypertensive patients with normal BMI. In overweight hypertensive
patients and overweight control subjects the plasma concentration of NOx was also lower, but
not significantly. These findings suggest that endothelial dysfunction, characterized by
decreased plasma NOx, may be more important pathogenetic mechanism in the essential
hypertension (EH) of lean patients than in obesity-induced hypertension (OH), but the
presence of endothelial dysfunction is also detectable in obesity and OH.
Conclusion 3. Increased plasma concentration of lipid peroxides, as malondialdehyde (MDA)
and a slight decrease in free thiol groups in overweight hypertrensive patients, elevated ratio
of oxidized/reduced glutathione (GSSG/GSH) due to decreased GSH and increased GSSG
concentrations in red blood cells and increased ratio of lipid peroxides/nitric oxide end-
products (MDA/NOx) in all hypertensive groups provide strong evidence of the presence of
oxidative stress in juvenile EH. In addition, the oxidative stress, characterized by MDA/NOx
ratio showed a significant correlation with BP. Oxidative stress seemed to be more
pronounced in overweight hypertensive patients than in hypertensive patients with normal
BMI. The decrease of plasma free thiols and increase of red blood cell GSSG in normotensive
overweight children allude to the presence of oxidative stress in obesity, irrespectively of
hypertension. Our data suggest that oxidative stress may be a pathogenetic factor in juvenile
EH, including OH. Obesity per se is a pro-oxidant state, and may aggravate oxidative stress,
if associated with hypertension.
43
Conclusion 4. In adolescents with EH the frequency of MT genotype of angiotensinogen
(AGT) gene M235T polymorphism was higher than in the normotensive subjects, but no
increase in the frequencies of the TT genotype and T allele has been detected. Therefore, the
presence of the T allele is not an obvious risk factor for EH. In addition, we failed to demon-
strate any other association between the gene polymorphisms of the renin-angiotensin system
(RAS) and juvenile EH. We conclude that gene variants of the RAS have no significant
influence on the development of EH in adolescents. Clarification of the exact role of these
gene polymorphisms in juvenile EH needs further investigations in wider patient population.
Conclusion 5. I/D polymorphism of the angiotensin-converting enzyme (ACE) gene and
M235T polymorphism of the AGT gene showed significant associations with pediatric end
stage renal disease (ESRD). The increased prevalence of the D allele and DD genotype was
demonstrated in this group of patients. The presence of the DD genotype could be a genetic
risk factor for renal parenchymal destruction, renal scarring, and the development of ESRD in
children, independent of the original renal disease. The MT genotype of AGT gene, which
was more frequent in pediatric ESRD, may contribute to the progression of CRF. These
genetic associations of pediatric ESRD were not established in adult patients.
Major findings of the thesis
I. The endothelial dysfunction may play a role in the pathogenesis of juvenile
essential hypertension, mainly in patients with normal body weight, but its
presence is also detectable in obesity and obesity-induced hypertension.
II. Increased oxidative stress state is observed in essential hypertension of adolescents,
irrespectively of the body weight. Obesity per se (without hypertension) is
associated with increased oxidative stress.
III. Gene polymorphisms of the renin-angiotensin system have no significant influence
on the development of essential hypertension in adolescents.
IV. Some gene polymorphisms of the renin-angiotension system (I/D polymorphism of
the ACE gene and M235T polymorphism of the angiotensinogen gene) may affect
the development of end stage renal disease in pediatric chronic kidney diseases.
44
8. ACKNOWLEDGEMENTS
I express my gratitude and thanks to Professor Sándor Túri, who introduced me to the
scientific and clinical fields of nephrology, guided my research activity, and supported me all
the way. I must really thank Dr. Csaba Bereczki for his guidance, scientific and clinical
advice and continuous support. I am truly grateful to Dr. Ibolya Haszon for the tremendous
help and encouragement over the years.
I would like to thank all my co-authors and co-workers who helped me in my laboratory
and clinical work, especially Dr. Emőke Endreffy, Dr. Ilona Németh, Dr. Eszter Karg, Dr.
Viktória Sümegi, Dr. Ákos Baráth, Dr. Péter Monostori, Dr. Gyula Wittmann, and Dr. Gábor
Rácz. I am grateful to the laboratory staff for their excellent technical assistance, especially to
Ildikó Csípő, Csilla Horváth, Ágota Fábiánné Nagy and Ilona Szécsi. I also greatly appreciate
the help of the staff of the Pediatric Dialysis Unit.
I am especially thankful for the support and useful advice of my colleague and friend
Dr. Balázs Gellén.
Without the persistent support, patience and help of my family, this work would not
have been possible.
45
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